Comparison of polymerase chain reaction single-strand conformation polymorphism with DNA sequencing to detect drug resistance of mycobacterium tuberculosis isolates

Advancements in molecular technology increased our understanding of genetic mechanism of drug resistance. Nowadays, the chance of rapid detection of resistant Mycobacterium tuberculosis [M. tuberculosis] strains is increased. In the present study, we aimed to investigate the sensitivity and specificity of PCR-SSCP for detecting susceptible and resistant strains of M. tuberculosis compared with DNA sequencing. To calculate the sensitivity and specificity of PCR-SSCP assay to detect drug resistance in M. tuberculosis, respiratory samples were collected from suspected patients referred to Mycobacteriology Research Center [Masieh Daneshvary Hosptial] since 2002. Susceptibility testing against first line drugs was performed on 74 culturepositive specimens. Consequently, PCR-SSCP and DNA sequencing were performed on katG, inhA, ahpC and rpoB genes. Drug-susceptibility testing by the proportional method in selected samples revealed 16 MDR [21.6%], 23 mono-drug resistant [31%] and 35 susceptible strains [47.3%]. In comparison with DNA sequencing as a gold standard for molecular methods, the sensitivity of PCR-SSCP assay for detecting of mutation in 315 codon of katG gene was 94.74% [CI=73.97%-99.87%] with 100% [CI=93.51%-100%] specificity. In contrast, the sensitivity and specificity of this assay in detecting of rpoB gene were 70.8% [CI=48.91%-87.38%] and 88% [CI=75.69%-95.47%], respectively. PCR-SSCP in combination with DNA sequencing can be used as screening method to detect MDR-TB and mono-drug resistant cases

Clonality of the immunoglobulin heavy chain genes in B cell non-Hodgkin lymphoma using semi-nested PCR

Identification of gene rearrangements and clonality analysis are important techniques for the diagnosis of malignant lymphoproliferative diseases. These methods have various sensitivities based on the type of primer used and method of determination of polymerase chain reaction [PCR] products. This study aimed at determining the clonality of B cell non-Hodgkin lymphoma in Iranian patients using PCR method and 2 primers of FR2 and FR3. Paraffin embedded blocks of 67 patients with B cell lymphoma and 19 cases with lymphoid hyperplasia of the lymph nodes who presented to NRITLD, Masih Daneshvari Hospital were retrospectively reviewed. After extracting the genomic DNA using phenol and chloroform, clonal analysis was performed using semi-nested PCR by using two primers: FR2 and FR3. PCR products were determined using 2 techniques of heteroduplex analysis, polyacrylamide gel and silver staining and the conventional method of agarose gel and ethidium bromide staining. Appearance of 1 or 2 bands in the desired location were considered as a sign of clonality. Monoclonal gene rearrangement was observed in 62 out of 67 patients [92.5%] as one or two discrete bands appeared within 60-120 base pairs [bp] and 200-300 bp range. Of the mentioned patients, 53 cases [79.1%] had FR2 and 51 [76.1%] had FR3 rearrangement. Heteroduplex analysis along with silver nitrate staining detected 3 out of the remaining 5 cases of lymphoma to be monoclonal. These cases had been reported negative by the conventional technique. In total, 65 out of 67 patients [97%] showed monoclonal gene rearrangement using both the abovementioned techniques. All hyperplasia cases were polyclonal by this method. Our study showed that evaluation and detection of clonality using PCR, FR2 and FR3 primers along with heteroduplex analysis is a rapid sensitive technique for the diagnosis of malignant lymphomas

clinicopathologic study of lung cancer cases in Iran

Lung cancer remains the leading cause of cancer-related deaths in the world. In Iran, lung cancer is the fifth leading cancer and its prevalence rate has been increasing steadily. In this study, the clinicopathological aspects of lung cancer are discussed. Between October 2002 and November 2005, 242 [178 men, 64 women] patients with histologically confirmed lung cancer were interviewed according to a questionnaire. Women developed the disease at an earlier age than men [55.9 +/- 14.2 versus 61.3 +/- 12.3 years; p=0.004]; 66.5% of lung cancer patients [85.4% of men and 14.1% of women] were smokers [p<0.0001]; 76.3% of participants, who had exposure to secondhand smoke, were females. Among the environmental carcinogens, the most exposures were to inorganic dusts [49.8%] and chemical compounds [34.9%].Most male and female patients suffered from adenocarcinoma [28.9%] and non small cell carcinoma [28.5%]. The prevalence of adenocarcinoma was higher in the non-smoker group, whereas incidence of squamous cell carcinoma and small cell carcinoma was higher among smokers [p<0.0001]. In this study, most patients [74.0%] presented with an advanced-stage tumor [IIIB or IV]. Our results suggest that in addition to cigarette smoking, other environmental, occupational and socioeconomic factors may play a role in the development of lung cancer

Insertion/deletion gene polymorphism and serum level of angiotensin converting enzyme

Angiotensin converting enzyme [ACE] plays an important role in cardiovascular regulation. Since the initial report regarding the association of insertion/deletion gene polymorphism and serum concentration of ACE, there have been investigations in different populations with contradicting results. The aim of this study was to assess the association of the ACE polymorphism and serum level in Iranians.The study recruited 88 healthy individuals [70 males and 18 females; mean age: 30.07 yrs.] who were candidates for kidney donation. To investigate the association of ACE serum level and polymorphism, the serum concentration of ACE was measured spectrophotometrically and ACE genotyping was determined by polymerase chain reaction.The genotype distribution of DD, ID and II was 31, 24 and 33 respectively. The mean ACE serum concentration for DD, ID and II genotypes was 50.68, 36.65 and 32.06 IU/L, respectively. There was a significant difference in ACE serum level among the three genotypes [p < 0.05]. While the highest ACE serum concentration was seen in DD group [nearly 1.5 times of that of ID and II genotypes, respectively], it was the lowest in the II group. Our study showed that insertion/deletion polymorphism of ACE gene was strongly associated with plasma ACE levels in the Iranian population

Achalasia as a potential predisposing factor for lung allograft rejection

Microaspiration secondary to gastroesophageal reflux has been postulated to be a predisposing factor for development of bronchiolitis obliterans syndrome after lung transplantation. Esophageal manometry and ambulatory pH monitoring have been suggested as a screening test in patients with end-stage lung disease. We report a single lung transplant patient who developed allograft rejection presumed to be due to underlying achalasia as the patient's clinical status and lung function improved markedly following the treatment of achalasia

Comparison of Nramp1 gene polymorphism among TB health care workers and recently infected cases; Assessment of host susceptibility

A link between polymorphisms in the natural resistance -associated macrophage protein gene 1 [Nramp] and susceptibility to tuberculosis [TB] has been demonstrated worldwide. This study aimed to investigate the Nramp1 gene variants among workers exposed to TB bacilli [1-2 hours per day for 1 to 20 years] who did not develop the diseases with those who developed the disease through recent transmission. The polymorphism of Nramp1 at INT4, D543 and 3'UTR was examined in 71 newly smear-positive TB cases and 39 healthcare workers exposed to TB. Polymerase chain reaction [PCR] and restriction fragment length polymorphism [RFLP] were used to genotype Nramp1 polymorphism. Patients' clinical and demographical data were collected. The heterozygote patterns of INT4 [G/C], D543 [G/A] and 3'UTR [+/del] occurred more frequently in control subjects than in patients [P =0.012], respectively [odds: 1.9 CI95%] [1.13-3.12]. Although, the homozygous patterns of INT4 [C/C; 8.5%], D543 [A/A; 1.4%] and 3'UTR [del/del; 1.4%] were only seen in patients [sensitivity 11% and specificity 100%]. The other risk factors like gender, age, resistance and PPD were not associated with Nramp1 gene polymorphism. Individuals with homozygous type mutation have an increased risk of developing tuberculosis. Therefore, we suggest detection of Nramp1 variants in high-risk groups i.e., health workers and close contact cases

unusual presentation of hodgkin's lymphoma as a chest wall abscess in association with old tuberculosis

Chest wall abscess is a very rare presentation of extranodal Hodgkin's lymphoma. Only a few case reports have been found in this regard. Here, we describe a chest wall mass developed in association with tuberculosis in an 82-year-old woman. Radiologic examination revealed two masses outside of chest wall that lead to destructive changes in the manubrium of sternum. The diagnosis of Hodgkin lymphoma was made by open surgical excisional biopsy of the chest wall mass. This is a very unusual extranodal presentation of Hodgkin's lymphoma

Assessment of angiotensin-converting enzyme gene in idiopathic pulmonary fibrosis

Interstitial pulmonary fibrosis [IPF] is a progressive fibrotic interstitial lung disease with a distinct histopathological form referred to as usual interstitial pneumonia [UIP]. Evidence has indicated that a local renin-angiotensin system is present in distal lung parenchyma. Expression of the component of this system is present in a number of fibrotic lung diseases. In this study, we assessed the association of Insertion/Deletion [I/D] polymorphism of angiotensin-converting enzyme [ACE] gene in IPF. By using semi-nested PCR, we determined the I/D polymorphism of ACE gene in 23 paraffinembedded open lung biopsy specimens from patients having clinical and imaging findings of IPF and pathologic diagnosis of UIP at National Research Institute of Tuberculosis and Lung Disease [NRITLD]. Afterwards, we compared the results with I/D polymorphism of ACE gene in a healthy control group [n= 88]. The frequency of I allele was 71.7%[33 out of 46] and the frequency of D allele was 28.3% [13 out of 46]. The frequent genotype was I/D [56.5%] which was statistically significant comparing with healthy group [27.3%]. We had no D/D genotype .There was a difference in the distribution pattern of ACE genotype between patients and controls [P < 0.05]. Our study revealed an association between carriage of I allele and I/D genotype in IPF

Fiberoptic bronchoscopy: correlation of cytology and biopsy results

Fiberoptic bronchoscopy is a diagnostic method for respiratory diseases. At present, its diagnostic yield has been increased by different cytologic and histologic procedures by convention. This study was conducted to evaluate the concordance and agreement between cytologic and histologic findings in conventional diagnostic bronchoscopic methods [washing and biopsy] for lung malignancies. This was a cross-sectional study performed on 2076 cases of bronchial biopsy and bronchial washing between 1996 and 2003. Of 2163 patients who underwent fiberoptic bronchoscopy after omitting 87[4%] cases due to unsatisfactory specimens, 2076 cases were studied including 832 [36.9%] females and 1244 [63.1%] males in the age range of 2 to 100 years, [mean age 57.7 +/- 16.3 yrs]. Male to female ratio was 1.5. Malignancy was diagnosed in 657[31.6%] biopsy and 283[13.6%] cytology specimens. Two hundred and sixty-five cases had malignant lesions according to both bronchial biopsy and bronchial washing; therefore, Kappa coefficient in both methods was 46.7% [P value = 0.000]. Concordance rate was 77.4%. Ninety-seven point three percent of malignant cases were diagnosed by biopsy and 41.9% by cytology. Cytology contributed to an additional diagnostic rate of 2.6%. Kappa agreement is classified as fair and although there is a very good concordance between the two sampling techniques, the diagnostic yield of cytology for malignancy must be improved by combination of multiple assays

TNF alpha gene polymorphism in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis [IPF] is characterized by a chronic inflammatory process and abnormal wound healing. Tumor Necrosis Factor alpha [TNF alpha] is considered to play a key role in fibroblast proliferation and increased collagen synthesis. It appears that there is a genetic predisposition to IPF. The genetic associations of TNF-alpha with IPF have been reported in different cohorts and revealing conflicting results. This study was conducted to evaluate the association of TNF- alpha-308 G/A polymorphism with IPF in Iranian patient by PCR-RFLP method. Materials and Methods: TNF alpha gene polymorphism at position 308 G/A was examined on DNA extract of 41 cases with IPF defined clinically, radiologically and histologically and compared with 82 unrelated healthy controls who were kidney donors. Results: The understudy population included 20 males and 21 females with the mean age of 50.4 yrs. Data showed that the frequencies of G allele [NO:72] and A allele [NO:10] were 87% and 12% respectively. The frequency of G/G genotype [NO:31] was 76%, followed by G/A [No:10] being 24%.we had no A/A genotype. Conclusion: There was no association between TNF alpha-308 G/A polymorphism and IPF in Iranian patients

Proliferative Marker in distinction between benign and malignant mesothelial proliferations

Differentiation of benign from malignant mesothelial proliferations is a major problem in the pathology of the serosal membranes, particularly in small biopsy specimen. This study was conducted for the evaluation of proliferative marker for distinction between malignant mesothelioma [MM] and mesothelial hyperplasia [MH]. Thirty six cases of malignant mesothelioma [MM] with the mean age of 62.94 years [range: 36-80 years, M/F: 3.58] and 22 cases of mesothelial hyperplasia [MH] were evaluated for proliferative status by immunohistochemical [IHC] method with monoclonal antibody, Ki-S5 [Ki-67]; the labeling indices [LI] were evaluated. Average count revealed a significant increase in MM as compared with reactive MH [p value < 0.0001]. Considering a threshold of 9% for ki-67, a sensitivity of 88% and specificity of 94% were resulted. Proliferative marker of Ki-67 can be useful in distinction between malignant mesothelioma and mesothelial hyperplasia [p-value < 0.0001]

Clinical pattern of idiopathic pulmonary fibrosis: a retrospective study

IPF is the most common form of interstitial lung disease. IPF is a clinico-pathological syndrome characterized by cough, exertional dyspnea, basilar crackles, a restrictive pattern on pulmonary function test [PFT] and honey comb pattern on HRCT. Since there are no exact data on IPF in Iranian patients and also the controversy that exist in this regard we decided to study the IPF cases in regard to epidemiologic, clinical features, radiologic manifestations and diagnosis in Masih Daneshvari Hospital during 1998-2001. This study was a descriptive retrospective study on files of IPF patients in Masih Daneshvari Hospital during 1998-2001. Although the total number of patients was 98, only 50 cases that had clinical and pathological findings compatible with IPF were included in the study. Twenty-seven [54%] were males and 23 [46%] were females. Mean age was 56.25 +/- 15.86 yrs. The most common clinical signs and symptoms were dyspnea [100%], cough [90%] and crackle [90%]. HRCT findings were abnormal in all patients. Eighteen percent were smokers and the most common occupational exposure was through agriculture. 82.2% of patients had restrictive pattern on PFT. Seventy percent of patients had transbronchial lung biopsy [TBLB], 26% had open lung biopsy [OLB] and 4% had video-assisted thoracoscopy [VATS]. Age of patients with IPF in our study is one decade lower than in Western countries. HRCT and TBLB assist significantly in the diagnosis of IPF and limiting the surgical procedures to only a limited number of cases

Detection of epstein barr virus DNA in thymic epithelial tumor using nested PCR

Thymus is a lymphoepithelial organ composed of epithelial cells and lymphocytes. Primary tumors of the thymus are uncommon and a definite risk factor has not been found. There are some reports regarding the association of the [EBV] Epstein Barr Virus with thymic epithelial tumors. This study was conducted to evaluate the presence of EBV genome in thymic epithelial tumor. EBV genome, EBNA2 was examined from DNA extracts of 41 paraffin embedded specimens including 16 thymic epithelial tumors as subject cases and 25 mediastinal lymph nodes as controls. Nested PCR assay revealed that 31.25% of cases were positive for EBV genome. The presence of EBV genome EBNA2 in thymic epithelial tumor suggesting that this association may be due to the endemic nature of EBV infection